Molecular Mechanism Of Severe Hemophilia Caused By Nonsense Mutation In F9Gene

Hemophilia is an X-linked recessive genetic disease that impairs the body’s ability to control blood clotting or coagulation and is caused by the mutation of coagulation factor gene (F8or F9). There are about80%severe patients with hemophilia. Individuals with severe hemophilia have functional coagulation factor levels that are less than1%of normal values and have frequent bleeding events, which are commonly associated with crippling arthropathy and early death. Restoration to～5%activity converts severe hemophilia to a mild form.According to the type of mutations registered in hemophilia database, we found that nonsense muations accounted to11.1%in F9gene mutations. To clarify the molecular mechanism of nonsense mutations leading to severe hemophilia, the NMD pathway and the NAS pathway in mammalian cells, the nonsense mutations in F9gene were identified and analysed to provide a theoretical basis for the treatment of genetic diseases caused by nonsense mutations. The following results are obtained:1. We constructed a mini-gene of Factor IX (Mini-hF9), which contains three parts:the first part contains the whole exon1and the5’ partial intron1(673bp); the second part contains the3’partial intron5, the whole exon6and the5’partial intron6(2115bp); and the last part contains the3’partial intron6, the whole exon7, intron7, and exon8(1553bp). The mini-gene was cloned into the mammalian expression vector pCMV-MH. According to RT-PCR and Western Blot analysis, the mini-gene could work in cells, product nomal transcripts and express full-length proteins.2. According to the type of mutations registered in hemophilia database, we designed and obtained the nonsense mutants E1(nt95T>A in Exon1), E7a (nt34G>T in Exon7), E7b (nt85G>T in Exon7), E7c (nt52G>T in Exon7) and E8(nt42C>T in Exon8) using PCR mutagenesis. RT-PCR analysis showed that two AS (alternative splicing) events (Alt-S1and Alt-S2) occurred in the nonsense mutants E7a, E7b and E7c. Western Blot analysis showed that there are the low levels of the C-terminal truncated protein in nonsense mutants E7a and E7c.3. The qRT-PCR results showed that the levels of the Norm mRNA decreased significantly in E7a and E7c mutants, and the levels of the Norm mRNA did not change in other mutants. The levels of the Norm mRNA increased significantly after CHX treatment in E7a and E7c mutants. In addition, we constructed the interference plasmids pSIR-hUPF1, pSIR-hSMG1, and the control plasmid pSIR-Control. Western Blot analysis indicated that the antisense hUPF1expression vector can decrease the endogenous UPF1protein levels and the antisense hSMG1expression vector can decrease the endogenous SMG1protein levels. qRT-PCR results showed that the levels of the Norm mRNA increased significantly after RNAi-hUPF1and RNAi-hSMG1treatment. These results indicate that the NMD pathway occurrs and decreases the Norm mRNA in E7a and E7c mutants.4. We used the miRNA algorithm TargetScan to predict and identify miRNAs that have the potential to regulate the NMD by trargeting the NMD factor SMG1. This algorithm identified miR-125a and miR-125b as potential regulators of SMG1. To determine whether miR-125directly targets the3’-UTR of SMG1, we amplified the500-nt-long3’-UTR of SMG1from human genomic DNA and cloned it into a dual-luciferase miRNA Target Expression Vector. Luciferase activity results showed that luciferase activity decreased after expression of miR-125, and luciferase activity increased after knock-down of endogenous miR-125. These data indicate that the predicted binding sites (miRNA response elements, MREs) in the seed sequence are critical for the direct and specific binding of miR-125to the SMG1mRNA.5. Western Blot analysis showed that the miR-125a or miR-125b could also decrease SMG1protein levels. The results indicate that miR-125 could regulate the expression of SMG1. In addition, qRT-PCR results showed that over-expression of miR-125a or miR-125b could decrease the SMG1mRNA levels; knock-down of endogenous miR-125a or miR-125b could increase the SMG1mRNA levels. The results indicate that miR-125down-regulates the SMG1protein levels by promoting its mRNA degradation.6. qRT-PCR analysis showed that over-expression of miR-125a or miR-125b could increase the NMD target mRNA levels; knock-down of endogenous miR-125a or miR-125b could decrease the NMD target mRNA levels. The results indicate that miR-125, as an NMD inhibitor, represses the NMD pathway by down-regulating the SMG1protein expression.7. According to the hypothesis from ESE Finder2.0, only E7a mutant might trigger alternative splicing by disrupting the function of the ESE sequence. We designed and generated three missense mutants, E7aE (nt34G>A in Exon7), E7bE (nt85G>A in Exon7) and E7cE (nt52G>C in Exon7), using PCR mutagenesis. RT-PCR analysis showed that AS events did not occur in the three missense mutants. The results indicate that the NAS pathway occurs and generates two AS events (Alt-S1and Alt-S2) in nonsense mutants E7a, E7b and E7c.8. To further verify our results, we determined the levels of the Norm and Alt-S1mRNA in cells transfected with an antisense hUPF2expression vector. We found that transfection of as-hUPF2increased the levels of the Norm mRNA expressed from the E7a or E7c mutant construct, demonstrating that this treatment effectively inhibited NMD. In contrast, as-hUPF2failed to significantly affect the levels of the Alt-S1mRNA, demonstrating that this treatment did not affect NAS. In addition, qRT-PCR analysis showed that the Alt-S1mRNA levels decreased significantly after transfection with as-hSMG6. The results indicate that SMG6might be an essential factor in the NAS pathway.9. qRT-PCR analysis showed that the proportion of the Norm, Alt-S1 and Alt-S2was respectively98.5%,1.1%and0.3%in WT (wild type); the proportion of the Norm, Alt-S1and Alt-S2was respectively73.7%,25.0%and1.4%in E7a mutant; the proportion was respectively92.2%,7.0%and0.8%in E7b mutant; the proportion was respectively68.1%,30.9%and1.1%in E7c mutant.10. We constructed the recombinant Mini-hF9gene and its stable nonsense mutant cell strains, HeLa-WT, HeLa-E7a and HeLa-E7c. RT-PCR and Western Blot analysis indicated that the constucts were successful. qRT-PCR analysis showed that the NMD target mRNA levels did not change after the treatment with different concentrations of promoting read-through drugs, PTC124and G418. The results indicate that PTC124and G418could not inhibit the NMD pathway.